Connective tissue growth factor (CTGF) is a growth factor induced in fibroblasts by many factors, including TGF-β, and is essential for the ability of TGF-β to induce anchorage-independent growth (AIG), a property of transformed cells. CTGF also is mitogenic and chemotactic for cells, and hence growth factors in this family are believed to play a role in the normal development, growth, and repair of human tissue. Five proteins related to CTGF, including Cyr61, Nov, WISP-1, WISP2, and WISP-3 have been isolated, cloned, sequenced, and characterized as belonging to the CCN gene family. Oemar and Luescher, Arterioscler. Thromb. Vasc. Biol., 17: 1483-1489 (1997); Brigstock, Endocrine Rev., 20:189-206 (1999). The gene encoding Cyr61 has been found to promote angiogenesis, tumor growth, and vascularization. Babic et al., Proc. Natl. Acad. Sci. USA, 95: 6355-6360 (1998). The nov gene is expressed in the kidney essentially at the embryonic stage, and alterations of nov expression, relative to the normal kidney, have been detected in both avian nephroblastomas and human Wilms' tumors. Martinerie et al., Oncogene, 9: 2729-2732 (1994). Wt1 downregulates human nov expression, which downregulation might represent a key element in normal and tumoral nephrogenesis. Martinerie et al., Oncogene, 12: 1479-1492 (1996).
The different members of the CCN family interact with various soluble or matrix associated macromolecules in particular sulfated glycoconjugates (Bork, FEBS Letters, 327:125-130). This interaction was used to purify Cyr61 and CTGF by affinity chromatography on heparin-agarose (Frazier et al., J. Invest. Dermatol., 107:404-411 (1996); Kireeva et al., Mol. Cell. Biol., 16:1326-1334 (1996)). Cyr61 is secreted and associated with both the extracellular matrix and the cell surface due to its affinity for heparan sulfate (Yang et al., Cell. Growth Diff., 2:351-357 (1991)). Recently, WISP-1 was shown to interact with decorin and biglycan, two secreted dermatan sulfate proteoglycans. (Desnoyers, et al., J. Biol. Chem., 276:47599-47607 (2001)).
The murine protein ELM1 was recently identified in low metastatic cells. Hashimoto et al., J. Exp. Med., 187:289-296 (1998). The elm1 gene, a mouse orthologue of WISP-1 disclosed below, is another member of the CNN gene family. It suppresses in vivo tumor growth and metastasis of K-1735 murine melanoma cells. Another recent article on rCop-1, the rat orthologue of WISP-2 described below, describes the loss of expression of this gene after cell transformation. Zhang et al., Mol. Cell. Biol., 18:6131-6141 (1998).
Wnts are encoded by a large gene family whose members have been found in round worms, insects, cartilaginous fish, and vertebrates. Holland et al., Dev. Suppl., 125-133 (1994). Wnts are thought to function in a variety of developmental and physiological processes since many diverse species have multiple conserved Wnt genes. McMahon, Trends Genet., 8: 236-242 (1992); Nusse and Varmus, Cell, 69: 1073-1087 (1992). Wnt genes encode secreted glycoproteins that are thought to function as paracrine or autocrine signals active in several primitive cell types. McMahon, supra (1992); Nusse and Varmus, supra (1992). The Wnt growth factor family includes more than ten genes identified in the mouse (Wnt-1, -2, -3A, -3B, -4, -5A, -5B, -6, -7A, -7B, -8A, -8B, -10B, -11, -12, and -13) (see, e.g., Gavin et al., Genes Dev., 4: 2319-2332 (1990); Lee et al., Proc. Natl. Acad. Sci. USA, 92: 2268-2272 (1995); Christiansen et al., Mech. Dev., 51: 341-350 (1995)) and at least nine genes identified in the human (Wnt-1, -2, -3, -5A, -7A, -7B, -8B, -10B, and -11) by cDNA cloning. See, e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534 (1984).
The Wnt-1 proto-oncogene (int-1) was originally identified from mammary tumors induced by mouse mammary tumor virus (MMTV) due to an insertion of viral DNA sequence. Nusse and Varmus, Cell, 31: 99-109 (1982). In adult mice, the expression level of Wnt-1 mRNA is detected only in the testis during later stages of sperm development. Wnt-1 protein is about 42 KDa and contains an amino-terminal hydrophobic region, which may function as a signal sequence for secretion (Nusse and Varmus, supra, 1992). The expression of Wnt-2 is detected in mouse fetal and adult tissues and its distribution does not overlap with the expression pattern for Wnt-1. Wnt-3 is associated with mouse mammary tumorigenesis. The expression of Wnt-3 in mouse embryos is detected in the neural tubes and in the limb buds. Wnt-5a transcripts are detected in the developing fore- and hind limbs at 9.5 through 14.5 days and highest levels are concentrated in apical ectoderm at the distal tip of limbs. Nusse and Varmus, supra (1992). Recently, a Wnt growth factor, termed Wnt-x, was described (WO95/17416) along with the detection of Wnt-x expression in bone tissues and in bone-derived cells. Also described was the role of Wnt-x in the maintenance of mature osteoblasts and the use of the Wnt-x growth factor as a therapeutic agent or in the development of other therapeutic agents to treat bone-related diseases.
Wnts may play a role in local cell signaling. Peifer and Polakis, Science, 287:1606-1609 (2000). Biochemical studies have shown that much of the secreted Wnt protein can be found associated with the cell surface or extracellular matrix rather than freely diffusible in the medium. Papkoff and Schryver, Mol. Cell. Biol., 10: 2723-2730 (1990); Bradley and Brown, EMBO J., 9: 1569-1575 (1990).
Studies of mutations in Wnt genes have indicated a role for Wnts in growth control and tissue patterning. In Drosophila, wingless (wg) encodes a Wnt-related gene (Rijsewik et al., Cell, 50: 649-657 (1987)) and wg mutations alter the pattern of embryonic ectoderm, neurogenesis, and imaginal disc outgrowth. Morata and Lawerence, Dev. Biol., 56: 227-240 (1977); Baker, Dev. Biol., 125: 96-108 (1988); Klingensmith and Nusse, Dev. Biol., 166: 396-414 (1994). In Caenorhabditis elegans, lin-44 encodes a Wnt homolog which is required for asymmetric cell divisions. Herman and Horvitz, Development, 120: 1035-1047 (1994). Knock-out mutations in mice have shown Wnts to be essential for brain development (McMahon and Bradley, Cell, 62: 1073-1085 (1990); Thomas and Cappechi, Nature, 346: 847-850 (1990)), and the outgrowth of embryonic primordia for kidney (Stark et al., Nature, 372: 679-683 (1994)), tail bud (Takada et al., Genes Dev., 8: 174-189 (1994)), and limb bud. Parr and McMahon, Nature, 374: 350-353 (1995). Overexpression of Wnt-1 in the mammary gland can result in mammary hyperplasia (McMahon, supra (1992); Nusse and Varmus, supra (1992)), precocious alveolar development (Bradbury et al., Dev. Biol., 170: 553-563 (1995)), and mammary adenocarcinomas (Li et al., Oncogene, 19:1002-1009 (2000)).
Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesoderm and the extraembryonic mesoderm of the day 7-8 murine embryo. Gavin et al., supra (1990). These embryonic domains contribute to the AGM region and yolk sac tissues from which multipotent hematopoietic precursors and HSCs are derived. Dzierzak and Medvinsky, Trends Genet., 11: 359-366 (1995); Zon et al., in Gluckman and Coulombel, ed., Colloque, INSERM, 235: 17-22 (1995), presented at the Joint International Workshop on Foetal and Neonatal Hematopoiesis and Mechanism of Bone Marrow Failure, Paris France, Apr. 3-6, 1995; Kanatsu and Nishikawa, Development, 122: 823-830 (1996). Wnt-5a, Wnt-10b, and other Wnts have been detected in limb buds, indicating possible roles in the development and patterning of the early bone microenvironment as shown for Wnt-7b. Gavin et al., supra (1990); Christiansen et al., Mech. Devel., 51: 341-350 (1995); Parr and McMahon, supra (1995).
For a review on Wnt, see Cadigan and Nusse, Genes & Dev., 11: 3286-3305 (1997).
Pennica et al., Proc. Natl. Acad. Sci., 95:14717-14722 (1998) describe the cloning and characterization of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, and a third related gene, WISP-3. (See also, WO 99/21998 published May 6, 1999; WO 99/21999 published May 6, 1999). Pennica et al. report that these WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis. WISP-1 has recently been identified as a β-catenin-regulated gene and the characterization of its oncogenic activity demonstrated that WISP-1 might contribute to β-catenin-mediated tumorigenesis (Xu et al., Gene & Develop., 14:585-595 (2000)). WISP-1 overexpression in normal rat kidney cells (NRK-49F) induced morphological transformation, accelerated cell growth and enhanced saturation density. In addition, these cells readily form tumors when injected into nude mice suggesting that WISP-1 may play some role in tumorigenesis (Xu et al., supra 2000). WISP-1 is also overexpressed in transformed human breast cancer cell lines and in about 47% of primary human breast cancer associated with certain advanced features. Xie et al., Cancer Res., 61:8917-8923 (2001); Saxena et al., Mol. Cell Biochem., 228:99-104 (2001); Michaelson et al., Oncogene, 20:5093-5099 (2001). A particular WISP-1 variant has also been reported to be overexpressed in about 86% of human scirrhous gastric carcinoma cells. Tanaka et al., Oncogene, 20:5525-5532 (2001).
Hurvitz et al., Nature Genetics, 23:94-97 (1999) describe a study involving WISP3 in which nine different mutations of WISP3 in unrelated individuals were found to be associated with the autosomal recessive skeletal disorder, progressive pseudorheumatoid dysplasia (PPD). WISP3 expression by RT-PCR was observed by Hurvitz et al. in human synoviocytes, articular cartilage chondrocytes, and bone-marrow-derived mesenchymal progenitor cells.
PCT application WO98/21236 published May 22, 1998 discloses a human connective tissue growth factor gene-3 (CTGF-3) encoding a 26 kD member of the growth factor superfamily. WO98/21236 discloses that the CTGF-3 amino acid sequence was deduced from a human osteoblast cDNA clone, and that CTGF-3 was expressed in multiple tissues like ovary, testis, heart, lung, skeletal muscle, adrenal medulla, adrenal cortex, thymus, prostate, small intestine and colon.
Hyaluronic acid (also referred to as HA, hyaluronate, or hyaluronan) is recognized in the literature as being an important component of the extracellular matrix (See, e.g., Hardingham et al., FASEB J., 6:861-870 (1992); Laurent et al., FASEB J., 6:2397-2404 (1992)). HA is a component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflamation, and embryonic morphogenesis. (Knudson et al., FASEB J., 7:1233-1241 (1993); Knudson et al., CIBA Found. Symp., 143:150-169 (1989)). HA has also been reported to play a role in certain types of metastases. (Naor et al., CD44: Structure, Function and Association with the Malignant Process, Advances in Cancer Research, Academic Press (1997), pages 241-319). The largest concentrations of HA are found in the skin and musculo-skeletal system which account for over 50% of total body HA. (Banerji et al., J. Cell Biol., 144:789-801 (1999)).
Various investigators have reported on receptors which bind HA. One of the receptors identified for HA is the CD44 protein. (See, e.g., Culty et al., J. Cell Biology, 111:2765-2774 (1990); Aruffo et al., Cell, 61:1303-1313 (1990); Naor et al., CD44: Structure, Function and Association with the Malignant Process, Advances in Cancer Research, Academic Press (1997), pages 241-319); Ropponen et al., Cancer Res., 58:342-347 (1998); Masaki et al., Cancer, 92:2539-2546 (2001). CD44 is a family of cell-surface glycoproteins generated from a single gene by alternative splicing and differential glycosylation. (Wielenga et al., Am. J. Pathology, 154:515-523 (1999)). CD44 is believed to function as a cell adhesion receptor, linking extracellular molecules, specifically hyaluronate, to the cell and the cytoskeleton (Wielenga et al., supra). CD44 is expressed on epithelial, mesenchymal and lymphoid cells. (Lesley et al., Adv. Immunol., 54:271-335 (1994)). Wielenga et al. report that CD44 expression may be regulated by the WNT pathway, based on certain experiments analyzing CD44 expression in the intestinal mucosa of mice and humans with genetic defects in either APC or Tcf-4. (Wielenga et al., supra).
Other HA receptors characterized to date include RHAMM (also referred to as receptor for hyaluronic acid mediated motility), a 58 kD intracellular protein expressed transiently on the surface of transformed lymphocytes (Hardwick et al., J. Cell Biol., 117:1343-1350 (1992); Turley et al., Exp. Cell Res., 207:277-282 (1993)). RHAMM expression in fibroblasts was reported to promote metastasis and play an important role in H-Ras transformation (Hall et al., infra).
Another receptor which binds HA was described by Banerji et al. (Banerji et al., supra). Banerji et al. report a receptor on lymph vessel walls, referred to as “LYVE-1”, which is a 322-residue type I integral membrane polypeptide which has a 41% similarity to the CD44 receptor. Unlike the CD44 receptor for HA, the LYVE-1 protein is absent in blood vessels. In addition, layilin (Bono et al., Mol. Biol. Cell, 12:891-900 (2001)) and HARE (Zhou et al., J. Biol. Chem., 275:37733-37741 (2000)) were also described as HA receptors.